臺灣博碩士論文加值系統

類澱粉乙型蛋白(Aβ)是組成阿茲海默症患者腦部老年斑塊且具有神經毒性的主要蛋白質，Aβ已知也會促使有絲分裂後已分化的神經元再進入細胞週期，進一步造成細胞死亡。許多研究指出植物萃取多醣體具有治療阿茲海默症的潛力，然而多醣體是藉由何種機制來阻止Aβ誘導的細胞死亡仍不清楚。因此，本篇研究旨在探討萃取自多年生藤本植物的多醣體(PPV-6)之神經保護機制。我們假設PPV-6會藉由阻斷Aβ所誘導的神經元再進入細胞週期來抑制神經細胞死亡。為了驗證這個假說，我們利用已分化之初代培養大腦皮質神經細胞共同處理PPV-6和Aβ，再利用西方點墨法、免疫細胞化學染色法與流式細胞儀來評估神經元再進入細胞週期的程度，此外也利用MTT試驗來分析細胞存活率。和單獨處理Aβ相比，PPV-6和Aβ共處理時不管是細胞存活率或是型態皆有回復。進一步的，處理PPV-6後Aβ所誘導的細胞週期標記蛋白質cyclin D1、pRb-Pi、PCNA、p-Histone H3與細胞凋亡指標cleaved caspase-3的上升皆會被回復，利用流式細胞儀也觀察到類似結果。總結來說，我們的研究指出PPV-6的神經保護機制是藉由阻止神經元再進入細胞週期，進而抑制細胞死亡。

Amyloid-beta peptide (Aβ) is the main neurotoxic component of senile plaque, which is the pathological hallmark of Alzheimer’s disease (AD). In addition, Aβ is also known to trigger cell cycle reentry in post-mitotic neurons followed by cell death. Many studies have reported that polysaccharides from medicinal plants may carry therapeutic potential in AD. However, the molecular mechanisms underlying polysaccharide-mediated inhibition of Aβ neurotoxicity still remain unclear. Therefore, this study was designed to explore the neuroprotective mechanisms of polysaccharides extracted from a perennial vine (PPV-6). We hypothesized that PPV-6 may suppress cell cycle reentry and subsequent apoptosis induced by Aβ in the fully differentiated post-mitotic neurons. To test this hypothesis, primary cortical neurons were subjected to co-treatment with Aβ and PPV-6. Western blotting, immunocytochemistry, and flow cytometry were conducted to assess the extents of neuronal cell cycle reentry. MTT assay was performed to determine cell viability. Compared with Aβ alone, cell viability and neuronal morphology were recovered by co-treatment with PPV-6. Further, Aβ-induced upregulation of G1-phase markers including cyclin D1 and phosphorylated retinoblastoma protein (pRb-Pi), G2-phase marker such as proliferating cell nuclear antigen (PCNA), mitotic marker histone H3 phosphorylated at Ser-10 (p-Histone H3), and caspase-3 cleavage indicative of apoptosis were all reversed by PPV-6. Similar results were obtained with flow cytometry. Taken together, our finding indicated that the neuroprotective mechanisms of PPV-6 involve, at least in part, suppression of Aβ-induced neuronal cell cycle reentry and subsequent apoptosis.

Contents

Acknowledgments ............................. i
Chinese abstract ............................ ii
English abstract ............................ iii
Contents .................................... iv
Introduction ................................ 1
Alzheimer’s disease (AD) ................... 1
AD and aberrant cell cycle reentry ......... 2
Polysaccharides and neurodegenerative diseases
............................................ 4
Materials and Methods ....................... 6
Reagents and preparations of Aβs ........... 6
Primary rat cortical cultures .............. 6
MTT reduction assay ........................ 7
Western blotting ........................... 7
Immunocytochemistry (ICC) .................. 8
Quantification of neurite lengths and neurite branches
............................................ 9
Flow cytometry ............................. 9
Statistical analysis ....................... 10
Results ..................................... 11
PPV-6 has neuroprotective effects against Aβ toxicity in
vitro ...................................... 11
PPV-6 restores Aβ25-35-induced neuronal morphology and
synaptic damage ............................ 11
PPV-6 decreases the numbers of the cell undergoing cell
cycle reentry induced by Aβ25-35 ........... 12
PPV-6 downregulates expression of cell cycle and apoptosis
markers in primary cortical cultures ....... 12
PPV-6 decreases the numbers of post-mitotic neurons
expressing cell cycle markers upon Aβ25-35 exposure
............................................ 13
PPV-6 reverses Aβ25-35-induced neuronal cell cycle reentry
in a post- treatment paradigm .............. 14
PPV-6 downregulates expression of cell cycle and apoptosis
markers induced by Aβ1-42 in primary cortical cultures
............................................ 15
Discussion .................................. 16
References .................................. 21
Figure legends .............................. 27

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